Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations

Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the “closure” of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and—more importantly—call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications.

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

“…The RECI region moves in the opposite direction with respect to RECIII and together with the R-rich helix, whereas the nuclease lobe remains stable (SI Appendix, Fig. S5) (7,13,24).…”

“…Finally, we also performed TMD simulations of Cas9:precat targeted to Cas9:DNA including all nucleic acid chains (i.e., RNA, t-DNA, and nt-DNA), revealing that the presence of the nt-DNA within the RuvC groove exerts a steric constraint that hampers the structural transition of HNH (φ does not change phase). This indicates that the transition of the HNH domain is unlikely to occur after complete DNA unwinding and relocation for the catalysis, suggesting that HNH repositioning might occur during the process of doublestrand separation (7,11,13).…”

“…dynamics (MD) simulations, we have recently portrayed the conformational dynamics of Cas9 as crystallized in the apo form and in complex with the nucleic acids (13). However, due to the short timescales accessible by conventional MD, this mechanistic study could not fully characterize the long-timescale conformational transitions arising from the interplay between protein and nucleic acids (12,14).…”

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…Structural studies suggested that a domain within the Cas9 recognition (REC) lobe (REC3) interacts with the RNA/DNA heteroduplex and undergoes conformational changes upon target binding (Extended Data Figure 2e–f) 13,14,17–19 . Because the function of this non-catalytic domain was previously unknown, we labeled SpCas9 with Cy3/Cy5 dyes at positions S701C (within the “mobile” REC3 domain) and S960C (within the “stationary” RuvC domain) to generate SpCas9 REC3 and observed that the conformational states of REC3 become more heterogeneous as PAM-distal mismatches increase (Extended Data Figure 4a–c).…”

“…Structural studies suggested that REC2 occludes the HNH domain from the scissile phosphate in the sgRNA-bound state 19 , and undergoes a large outward rotation upon binding to double-stranded DNA (dsDNA) 13,14 (Figure 2e). To test whether the REC2 domain regulates access of HNH to the target strand scissile phosphate, we labeled SpCas9 with Cy3/Cy5 dyes at positions E60C (within the “stationary” Arginine-rich helix) and D273C (within the “mobile” REC2 domain) to generate SpCas9 REC2 in order to detect REC2 conformational changes (Extended Data Figure 1b–c).…”